Synaptic plasticity, the cellular correlate to learning and memory, is mediated by highly regulated signaling cascades, compartmentalized in small dendritic spines. The regulation in space and time of the hundreds of proteins involved in these cascades enables short-lived synaptic inputs to be transduced into long-lasting structural and functional synaptic modifications. The protein kinase C (PKC) family, consisting of more than 12 isozymes, has been implicated to play an essential role in the induction, expression and maintenance of synaptic plasticity. However, limitations in current experimental approaches, including poor isozyme discrimination, spatiotemporal resolution, and sensitivity, have limited understanding of the precise role of PKC isozymes in the signaling cascades that mediate these changes. To overcome these problems, the first aim of this study is the development of new, highly-optimized, fluorescence-based sensors for PKC isozymes based on fluorescence resonance energy transfer (FRET) and 2-photon fluorescence lifetime imaging (2pFLIM). These highly sensitive sensors will report isozyme-specific PKC activity with submicron spatial and subsecond temporal resolution upon stimulation of a single spine. Taking advantage of multiple activation steps of PKC isozymes, several sensors will be developed for each of eight PKC isozymes shown to have a role in spine plasticity. Through the use of these sensors, 2pFLIM, and glutamate uncaging, the spatiotemporal profile of isozyme-specific PKC activity during single spine structural plasticity will be elucidated in aim two of the proposed study. The requirement of specific isozymes in the induction, expression and maintenance of structural plasticity will then be examined by inhibition of specific isozymes. Finally, the upstream activation of PKC isozymes will be examined to determine the requirement of receptor activation for plasticity, as well as the heterogeneity of isozyme activity to various inputs. The experiments proposed in this study will provide insight into how PKC fits into the complex signaling networks that mediate plasticity. This will enhance understanding of the molecular mechanisms of synaptic plasticity; the dysfunction of which is a feature of many neuropsychiatric disorders. In particular these studies may provide insight into disorders such as Alzheimer's disease and bipolar disorder, in which PKC dysfunction has been implicated. Finally, since PKC is involved in the regulation of numerous cell processes, the tools developed here will be useful for the broader cell biological community, including the study of other diseases related to PKC function such as cancer.